Composition and method for the detection of diseases associated with amyloid-like fibril or protein aggregate formation

ABSTRACT

The present invention relates to novel compositions useful for elucidating the onset or progress of diseases such as Huntington&#39;s disease, that are associated with the formation of fibrils or protein aggregates. Further, the present invention relates to methods for monitoring formation of fibrils or protein aggregates as well as to methods for identifying inhibitors of fibril or protein aggregate formation. Additionally, the invention relates to inhibitors of the formation of fibrils or protein aggregates identified by the method of the invention as well as to pharmaceutical compositions that include the inhibitors.

RELATED APPLICATIONS

This application claims priority to PCT/EP98/04811, filed on Jul. 31,1998, which claims priority to European Patent Application 97113306.1,filed on Aug. 1, 1997.

FIELD OF THE INVENTION

The present invention relates to novel compositions useful forelucidating the onset or progress of diseases of preferably neuronalorigin associated with the formation of amyloid-like fibrils or proteinaggregates. Further, the present invention relates to methods formonitoring said formation as well as to methods for identifyinginhibitors of said formation. Additionally, the invention relates toinhibitors identified by the method of the invention as well as topharmaceutical compositions comprising said inhibitors.

BACKGROUND OF THE INVENTION

A variety of diseases, both in humans and animals, is characterized bythe pathogenic formation of amyloid-like fibrils or protein aggregatesin neuronal tissues. A well-known and typical example of such diseasesis Alzheimer's disease (AD). AD is characterized by the formation ofneurofibrillar tangles and β-amyloid fibrils in the brain of ADpatients. Similarly, scrapie is associated with the occurrence ofscrapie-associated fibrils in brain tissue.

Another class of these diseases is characterized by an expansion of CAGrepeats in certain genes. The affected proteins display a correspondingpolyglutamine expansion. Said diseases are further characterized by alate onset in life and a dominant pathway of inheritance.

A typical representative of this class of diseases is Huntington'sdisease. Huntington's disease (HD) is an autosomal dominant progressiveneurondegenerative disorder characterized by personality changes, motorimpairment and subcortical dementia (Harper, 1991). It is associatedwith a selective neuronal cell death occurring primarily in the cortexand striatum (Vonsattel et al., 1985). The disorder is caused by aCAG/polyglutamine (polygln) repeat expansion in the first exon of a geneencoding a large ˜350 kDa protein of unknown function and designatedhuntingtin (HDCRG, 1993). The CAG repeat is highly polymorphic andvaries from 6–39 repeats on chromosomes of unaffected individuals and35–180 repeats on HD chromosomes (Rubinsztein et al., 1996; Sathasivamet al., 1997). The majority of adult onset cases have expansions rangingfrom 40–55 units, whereas expansions of 70 and above invariably causethe juvenile form of the disease. The normal and mutant forms ofhuntingtin have been shown to be expressed at similar levels in thecentral nervous system and in peripheral tissues (Trottier et al.,1995a). Within the brain, huntingtin was found predominantly in neuronsand was present in cell bodies, dentrites and also in the nerveterminals. Immunohistochemistry, electron microscopy and subcellularfractionations have shown that huntingtin is primarily a cylosolicprotein associated with vesicles and/or microtubules, suggesting that itplays a functional role in cytoskeletal anchoring or transport ofvesicles (DiFiglia et al., 1995; Gutekunst et al., 1995; Sharp et al.,1995) Huntingtin has also been detected in the nucleus (de Rooij et al.,1996; Hoogeveen et al., 1993) suggesting that transcriptional regulationcannot be ruled out as a possible function of this protein.

In addition to HD, CAG/polygln expansions have been found in at leastseven other inherited neurodegenerative disorders which include: spinaland bulbar muscular atrophy (SBMA), dentatorubral pallidoluysian atrophy(DRPLA), and the spinocerebellar ataxias (SCA) types 1, 2, 3, 6 and 7(referenced in Bates et al. 1997). The normal and expanded size rangesare comparable with the exception of SCA6 in which the expanded allelesare smaller and the mutation is likely to act by a different route.However, in all cases the CAG repeat is located within the coding regionand is translated into a stretch of polygln residues. Although theproteins harbouring the polygln sequences are unrelated and mostly ofunknown function, it is likely that the mutations act through a similarmechanism. Without exception, these proteins are widely expressed andgenerally localized in the cytoplasm. However, despite overlappingexpression patterns in brain, the neuronal cell death is relativelyspecific and can differ markedly (Ross, 1995), indicating thatadditional factors are needed to convey the specific patterns ofneurodegeneration.

Several investigators have proposed that HD is caused by a toxic gain offunction, which in turn is caused by abnormal protein—proteininteractions related to the elongated polygln. It is possible that thebinding of a protein to the polygln region could either confer a newproperty on huntingtin or alter its normal interactions causingselective cell death either through the specific expression patterns ofthe interacting protein or through the selective vulnerability ofcertain cells. To date, four potential huntingtin-interacting proteinshave been isolated: 1 (Li et al., 1995), GAPDH (Burke et al., 1996),HIP2 (Kalchman et al., 1996) and HIP-I (Kalchman et al.,: 1997; Wankeret al., 1997). However, it has not been demonstrated whether the bindingof these proteins to huntingtin is involved in the selectiveneuropathology. A gain of function mechanism has been supported by theidentification of an antibody that specifically reacts with thepathogenic polygln expansions (Trottier et al., 1995b) This indicatesthat upon expansion into the pathogenic range, a polygln sequence mayundergo a conformational change. Poly-L-glutamines form pleated sheetsof β-strands held together by hydrogen bonds between their amides(Perutz et al., 1994). It was proposed that the expanded glutaminerepeats in huntingtin may function as polar zippers, joining proteinmolecules together (Perutz, 1996). In the long run, this could result inthe precipitation of huntingtin protein in specific neurons causing theobserved selective neuronal loss. Thus, the mechanism underlying HDwould be similar to scrapie, Creutzfeldt-Jakob or Alzheimer's disease,in which β-sheet secondary structures lead to the formation of toxicprotein aggregates in selective neurons (Caughey and Chesebro, 1997).

Recently, strains of mice (R6) that are transgenic for the HD mutationhave been generated (Mangiarini et al., 1996). In these mice exon 1 ofthe human HD gene carrying CAG repeat expansions of 115–156 units isexpressed under the control of the human HD promoter. It has beendemonstrated that the transgenic animals exhibit a progressiveneurological phenotype that exhibits many of the motor and non motorfeatures of HD. The phenotype includes a resting tremor; irregular gait;rapid, abrupt shuddering movements; stereotypic grooming movements andepileptic seizures. Coincident with the onset of the movement disorderthe mice show a progressive weight loss. Neuropathological analysis hasshown a reduction in brain weight (which precedes that in body weight)and the presence of neuronal intranuclear inclusions (NIIs) predatingany evidence of neuronal dysfunction (Davies et al., 1997). The NIIs areimmunoreactive for N-terminal huntingtin antibodies that detect thetransgene protein and for ubiquitin but do not contain the endogenousmouse huntingtin. At the ultrastructural level, a solitary intranuclearinclusion appears as a roughly circular pale structure of a finegranular nature with occasional filamentous structures and devoid of amembrane. In addition, the neurons invariably have indentations of thenuclear membrane and an apparent increase in the density and clusteringof nuclear pores. All three of these ultrastructural nuclear changeshave previously been reported in EM studies from HD patients (Roizin etal., 1979; Roos and Bots, 1983; Tellez-Nagel et al., 1974).

Thus, a large body of data has accumulated that describes aspects of thepathology of the above-discussed diseases. However, the actualmechanisms leading to the onset of the various disease states are stillunknown. Although a variety of hypotheses have been formulated in theart, it is equally unknown how the amyloid or aggregate formation istriggered or caused within affected cells or tissues. Without a detailedknowledge of the formation of said aggregates, the development of asuitable pharmaceutical composition for treating such diseases appearsrather difficult. The technical problem underlying the present inventionwas therefore to provide means and methods suitable for the eventualelucidation of the etiology of these diseases and the development ofappropriate medicines.

SUMMARY OF THE INVENTION

The above technical problem is solved by the embodiment characterized inthe claims. Accordingly, the present invention relates to a compositioncomprising:

-   -   (a) a nucleic acid molecule encoding a fusion protein        comprising:    -   (aa) a (poly)peptide that enhances solubility and/or prevents        aggregation of said fusion protein; and    -   (ab) an amyloidogenic (poly)peptide that has the ability to        self-assemble into amyloid-like fibrils or protein aggregates;    -   (b) a vector containing the nucleic acid molecule of (a);    -   (c) a host transformed with the vector of (b);    -   (d) a fusion protein encoded by the nucleic acid of (a) or a        functional derivative thereof; and/or    -   (e) an antibody specific for the fusion protein of (d).

As used herein, the term “(poly)peptide” relates to a polypeptide or apeptide depending on the length of the amino acid string. Said(poly)peptide has the ability to enhance solubility of a fusion partnerin said fusion protein and thus of the fusion protein itself.Additionally, or alternatively, said (poly)peptide prevents theaggregation of the fusion partner and thus of the fusion protein. Said(poly)peptide is combined within said fusion protein with anamyloidogenic (poly)peptide having the above recited features. Theconnection of both (poly)peptides may be via a linker or by a directattachment. It is preferred that either the linker or either(poly)peptide comprises a cleavable site. Said cleavable site shouldrender both (poly)peptides essentially intact. Alternatively, saidfusion protein may comprise a number of cleavage sites. Upon cleavage,which may be exhaustive or under limiting conditions, the amyloidogenic(poly)peptide should, when used for the purposes of the presentinvention, retain the ability to self-assemble. The person skilled inthe art is in the position to determine appropriate conditions for acorresponding limited cleavage. The composition of the invention maycomprise one, several, or all of the compounds recited in features (a)to (e), above.

The term “functional derivative” refers to a fusion protein whichcomprises, for example, modified amino acids or amino acid substitutionsand retains the functions of the fusion protein detailed herein above.

The term “antibody specific for the fusion protein” comprised in thecomposition of the invention is intended to mean that said antibody isonly specific for the fusion protein but not for either of the abovecited components of said fusion protein.

In accordance with the present invention, it could surprisingly be shownthat the composition comprising the above recited components can be usedfor the elucidation of amyloid-like fibril or protein aggregateformation. The components of the composition can be used in varyingcombinations to test, for example, for specific conditions under whichamyloids are formed in vitro. The in vitro data obtained with thecomposition of the invention may then be compared to or brought intorelation with the in vivo situation and appropriate conclusions may bedrawn therefrom.

The in vitro systems that can be established with the composition of theinvention allow formation of highly stable amyloid-like proteinaggregates. Such aggregates may be obtained, for example, by proteolyticcleavage of GST fusion proteins comprising exon 1 of the HD gene andcontaining expanded polygln sequences. Alternatively, such aggregatesmay be obtained by lowering the pH value from 8 to 5 or by increasingthe protein concentration. The arrays of fibrillar structures of varyingsizes and shapes observed by electron microscopy surprisingly clearlyresemble those of purified amyloids. Furthermore, the polarizationmicroscopic properties of the fibrils stained with Congo red arestrikingly similar to those described for amyloids. The green-goldbirefringence of the amyloid-like fibrils indicates that the polymershave common structural features. Although the Congo red staining doesnot determine conclusively whether the fibrils consist of β-pleatedsheets, the method suggests that this is likely in view of experiencegained with other protein polymers (Caputo et al., 1992). However, ithas been generally accepted that naturally occurring mammalian proteinpolymers that exhibit fibrillar structures and green birefringence afterCongo red staining should be classified as amyloids (Glenner, 1980).Instead of the HD gene, other nucleic acid sequences encodingamyloidogenic (poly)peptides may be used to generate said fusionproteins. Preferably, the composition of the invention is a diagnosticcomposition. The composition of the invention may also be a kit. Thediagnostic composition can advantageously be employed in the assessmentof a disease state whereas the kit may rather be employed in thedevelopment of, for example, inhibitors or in the elucidation of amyloidformation.

It is a preferred embodiment of the composition of the invention, thatsaid amyloidogenic (poly)peptide comprises a polyglutamine expansion. Inthe prior art it has been shown by X-ray diffraction studies thatsynthetic peptides containing polyglns form β-sheets strongly heldtogether by hydrogen bonds (Perutz et al., 1994). Because syntheticpoly(L-glutamine) is insoluble in water, a synthetic peptide with thesequence Asp₂-Gln₁₅-Lys₂ was used in that study. A stretch of 10glutamines was also inserted into the loop of chymotrypsin inhibitor-2(Cl2), and it was demonstrated by analytical ultracentrifugation thatthe recombinant protein, in addition to monomers formed dimers andtrimers, whereas wild-type Cl2 was present only in the monomeric form(Stott et al., 1995). It has been proposed that the polygln stretchfunctions as a polar zipper, joining proteins together. However, thehypothesis that glutamine repeats in proteins form β-pleated sheets andinduce protein aggregation by a mechanism similar to that observed inspongiform encephalopathy (TSE) diseases (Caughey and Chesebro, 1997)could not be proven with this recombinant protein. Most likely, thelength of the polygln sequence inserted into Cl2 was too short.Accordingly, the experimental data actually obtained teach away from theabove recited hypothesis.

In a particularly preferred embodiment said polyglutamine expansioncomprises at least 35 glutamines. In a further particularly preferredembodiment said polyglutamine expansion comprises at least 51glutamines.

Our studies with the GST-HD fusion proteins containing polygln sequencesof varying lengths demonstrate that a certain length of the polyglnstretch is necessary for the formation of amyloid-like fibrils in vitro.When the purified fusion proteins were analyzed by SDS-PAGE, insolublehigh molecular weight protein aggregates were only detected with theproteins containing 81 and 122 glutamines (FIGS. 1 a and b), whereas theprotein with 51 glutamines was soluble and no fibrillar structures weredetected by electron microscopy (FIG. 4 a). This indicates that thecritical length of the polygln stretch in the fusion proteins leading tothe formation of aggregates is greater than 51 glutamines. Accordingly,fusion proteins of the invention with a polyglutamine expansion of morethan 51 glutamines, such as 81 or 122 glutamines, may be employed instudies for the formation of aggregates that render a cleavage reactionunnecessary. However, when the GST-tag, which is known to enhance thesolubility of many proteins (Smith and Johnson, 1988), was cleaved bylimited digestion with trypsin, the liberated HD exon 1 protein with 51glutamines also started to form aggregates (FIG. 3 a) and the amount ofthese aggregates increased when the GST-tag was totally degraded withtrypsin (FIG. 3 b). This indicates that in the HD exon 1 protein 51glutamines are sufficient to form aggregates, whereas 20 and 30glutamines under the same conditions are not. The minimum criticallength essential for the development of amyloid-like structures afterremoval of the GST-tag is not known and has to be determined. However,preliminary experiments in our laboratory suggest that the threshold forthe formation of HD exon 1 protein aggregates is between 35–48glutamines. This result is strikingly similar to the pathologicalthreshold in HD, SBMA, DRPLA, SCA1, SCA2, SCA3 and SCA7. In all of theseneurodegenerative polygln diseases a pathological phenotype was foundwhen more than 41 repeats were present, suggesting that the elongationof the polygln repeat beyond a certain length may lead to a phase changein the affected proteins. This could, for example, be a change fromrandom coils to hydrogen-bonded hairpins in the polygln stretch, seePerutz (1996).

With the understanding that the applicant is not bound by any scientifictheory, a mechanism is proposed for the fibril formation induced byproteolytic: cleavage of GST-HD51 as shown in FIG. 7. Based on the knowncrystal structure of GST with a C-terminal fusion peptide (Lim et al.,1994) and the fact that the purified GST protein is a dimer we supposethat native GST-HD51 exists as a dimer with two expanded polyglnsequences which form stable hairpins consisting of antiparallelβ-strands strongly held together by hydrogen bonds between the mainchain and the side chain amides. In the native protein both hairpins aretightly bound to the surface of GST and not accessible forprotein—protein interactions with other polygln sequences. As a resultof the cleavage with a site-specific protease, both hairpins becomeaccessible and β-sheets with hairpins from other cleaved proteinmolecules are formed. This transient population of intermediatesconsisting of GST molecules and hairpins leads to the formation ofpolygln-containing β-sheet fibrils and free GST molecules. This model issupported by the finding of potential intermediate structures present onone or both ends of the growing fibrils (FIGS. 4 c and d). These clotsof varying sizes were not detected when GST-HD51 was digested tocompletion with trypsin, which totally degrades the GST-tag, whilst theywere detectable upon limited digestion, leaving the GST moiety largelyintact (FIG. 4 d). This indicates that these structures are transientintermediates.

A model of the formation of amyloid-like fibrils via transientintermediates is not without precedent. Booth et al. (1997) have shownthat amyloidogenic lysosome variants aggregate on heating, unlike thewild-type protein, and that the lysozyme fibrils are formed frompotential precursor proteins. It is possible that the transient GST-HDintermediates function as nuclei for ordered protein aggregation, verysimilarly to protein crystallization and microtubule formation, whichare nucleation-dependent polymerisations (Jarrett and Lansburry, 1993).Once a nucleus is formed, the further addition of monomers becomesthermodynamically favorable and results in rapid polymerization. Animportant feature of a nucleation-dependent process is a lag time beforethe aggregates are detectable. During this period, dimers and trimersare formed. FIG. 3 a shows that during proteolytic cleavage of GST-HD51dimers of the released HD portion are formed, the concentration of whichthen decreases upon prolonged incubation concomitant with an increase inthe formation of large protein aggregates (FIG. 3 b). Althoughadditional kinetic studies will be necessary to prove this assumption,preliminary results in our laboratory suggest that a “one-dimensional”crystallization leads to the formation of in vitro amyloid-likehuntingtin aggregates.

Accordingly, the present invention provides both the possibilities toanalyze aggregation of amyloid-like aggregates using defined cleavageconditions or using fusion proteins comprising long polyglutaminestretches that render the cleavage unnecessary. Whereas the cleavage ofthe fusion protein enables to set a distinct starting point of thereaction and therefore of the aggregate formation, the secondalternative has the advantage that the use of a cleaving agent isrendered obsolete.

In a further particularly preferred embodiment of the invention said(poly)peptide defined in (ab) is huntingtin, androgen receptor, atropin,TATA binding protein, or ataxin-1, -2, -3, -6 or -7 or a fragment orderivative thereof.

The fibrillar structures formed by proteolytic cleavage of purifiedGST-HD51 in vitro and also in the brains of mice transgenic for the HDmutation are very similar to structures detected in brain sections orpurified protein fractions of Alzheimer's disease (AD),Creutzfeldt-Jakob disease (CJD), Parkinson's Disease,Gerstmann-Strätssler-Scheinker syndrome (GSS), fatal familial isomnia(FFI), kuru, bovine spongiform encephalopathy (BSE) and scrapie (Caugheyand Chesebro, 1997). In all these disorders, the accumulation ofamyloid-like fibrils in the central nervous system is accompanied byloss of nerve cells and a neuropathological phenotype. However, themolecular basis of these diseases is not known. For the first time, ourresults raise the possibility that HD, DRPLA, SBMA, SCA1, SCA2, SCA3 andSCA7 are also the result of a toxic amyloid fibrillogenesis. Althoughthe detection of amyloid-like fibrils has not previously been reportedin these inherited diseases, our results strongly suggest thatpolygln-containing polymers are also formed in vivo by their detectionin a transgenic model of polyglutamine disease. The high molecularweight aggregates were exclusively detected in the nuclear fractionprepared from transgene brain material, which is in good agreement withthe results of Davies et al. (1997), who demonstrated the presence ofthe transgene protein and ubiquitin in neuronal intranuclear inclusions,from a time prior to the development of a neurological phenotype.Strikingly, ultrastuctural analysis has shown similar intranuclearinclusions to be present in the cortical and striatal biopsy materialfrom HD patients (Roizin et al., 1979) some of which showed clearevidence of intranuclear fibrils of up to 1 μm in length. Preliminaryexperiments with nuclear protein fractions prepared from HD brainmaterial indicated that insoluble huntingtin aggregates are indeedpresent in these fractions. However, additional control experiments haveto be performed to substantiate these results.

One possible explanation for the absence of detection of high molecularweight huntingtin protein aggregates in HD brains could be that theaggregates consist mainly of polygln-containing peptides which have beencleaved from the full length protein. In such a case, only an antibodyraised against an N-terminal huntingtin fragment, containing the polyglnsequence, would be able to detect the aggregates in the nucleus. In mostof the previous immunohistochemical studies, antibodies raised againstthe central or C-terminal portion of huntingtin have been used, whichdetect the full length protein (350 kDa) in the cytosol and in themembrane containing fractions (DiFiglia et al., 1995; Sharp et al.,1995; Trottier et al., 1995a). However, antibodies raised againstpeptides and fusion proteins from the N- and C-terminus of huntingtinalso detected the protein in the nucleus (de Rooij et al., 1996;Hoogeveen et al., 1993), indicating that huntingtin is also present inthis subcellular compartment. There are several lines of evidence toimplicate a shorter polygln-containing peptide/protein fragment ofhuntingtin in the pathology of HD. Ikeda et al. (1996) showed that ashort fragment of the MJD1 protein containing 79 polyglns (Q79C) but notthe full length protein with the elongated repeat induced apoptotic celldeath in COS cells. The polygln-containing protein fragment migrated inSDS-gels at a position much higher than expected from its molecularweight, even after boiling in the presence of 2% SDS. These results arein good agreement with our data obtained using the GST-HD fusionproteins containing elongated polygln sequences. FIGS. 1 a and b showthat the expression of GST-HD83 and GST-HD122 in E. coli wasdramatically reduced compared to the fusion proteins containing 20–51repeats, and additional studies have indicated that the elongatedglutamines are toxic for E. coli cells.

The possibility that polygln-containing cleavage products of huntingtincause neurodegeneration in HD is substantiated by the finding ofGoldberg et al. (1996) who showed that an N-terminal 80 kDa huntingtinfragment is cleaved from the full length protein by apopain, aproapoptotic cysteine protease. This indicates that the N-terminus ofhuntingtin is primarily accessible for proteases and distinctproteolytic cleavage products can be formed in vitro and in vivo. Inaddition, there is strong evidence that the mutated huntingtin somehowinduces apoptotic cell death in HD, but the underlying molecularmechanism is not known (Duyao et al., 1995; Portera-Cailliau et al.,1995). Our data suggest that a proteolytic cleavage product ofhuntingtin, which is transported into the nucleus by an unknownmechanism, causes selective neuronal cell death by the formation ofinsoluble amyloid-like fibrils. It is possible that the transport to thenucleus is facilitated by a specific nuclear transport mechanism whichis unique to certain neuronal cells and involves abnormalprotein—protein interactions related to the elongated polygln.Alternatively, there may be specific nuclear proteins in the affectedneurons which enhance the huntingtin protein aggregation.

Recently, the formation of neuronal intranuclear inclusions in micetransgenic for the SCA1 mutation have been detected, indicating thatpolygln-containing polymers are also formed in spinocerebellar ataxiatype 1. Furthermore, the accumulation of polyglutamine-containingprotein aggregates in neuronal intranuclear inclusions (NIIs) has beendemonstrated for several progressive neurodegenerative diseases such asHuntington's disease (HD) (M. DiFiglia et al., 1997; M. W. Becher etal., 1997), dentatorubral pallidoluysian atrophy (DRPLA) (M. W. Becheret al., 1997; S. Igarashi et al., 1998) and the spinocerebellar ataxia(SCA) types 1 (P. J. Skinner et al., 1997; A. Matilla et al., 1997), 3(H. L. Paulson et al., 1997) and 7 (M. Holmberg et al., 1998).

The components of the composition of the invention may be packaged incontainers such as vials, optionally in buffers and/or solutions. Ifappropriate, one or more of said components may be packaged in one andthe same container.

In an additional preferred embodiment of the composition of the presentinvention, said amyloidogenic (poly)peptide self-assembles subsequent torelease from said fusion protein.

As has been pointed out herein before, the self-assembly of said(poly)peptides only subsequent to the release from the fusion proteinprovides the advantage that an exact time point of the start of theformation can be set. This has a number of advantages. For example,inhibitors of aggregate formation can be tested as regards theirefficacy as a function of time. In addition, obtainment of data isfacilitated in view of the fact that for amyloid formation a premix maybe set up to which only the cleaving agent must be added.

In a further preferred embodiment of the composition of the invention,said amyloidogenic (poly)peptide is the amyloid precursor protein (APP),β-protein, an immunoglobulin light chain, serum amyloid A,transthyretin, cystatin C, β2-microglobulin, apolipoprotein A-1,gelsoline, islet amyloid polypeptide (IAPP), calcitonin, a prion, atrialnatriuretic factor (ANF), lysozyme, insulin, fibrinogen, tau proteins orα-synuclein or a fragment or derivative thereof.

Deposits of β-amyloid in neuritic plaques and blood vessel walls are theprincipal pathological feature in the brains of patients withAlzheimer's disease. These amyloid deposits contain the 39–43 amino acidβ-amyloid peptide which is derived by proteolytic cleavage from thelarger precursor, the amyloid precursor protein (APP). There is strongevidence that the formation and aggregation of β-amyloids into fibrilsis the primary pathogenic event leading to amyloid deposition inAlzheimer's disease.

An additional preferred embodiment relates to a composition wherein said(poly)peptide defined in (aa) is glutathione S-transferase (GST),intein, thioredoxin, dihydrofolate reductase (DHFR) or chymotrypsininhibitor 2 (Cl2) or a functional fragment or derivative thereof.

All of these (poly)peptides may be advantageously used to enhance thesolubility and/or prevent aggregation of the fusion proteins of theinvention. Particularly preferred is to employ intein in saidcomposition because the protein has been modified such that it undergoesa self-cleavage reaction at its N-terminus at low temperatures in thepresence of thiols such as DTT (MPACT™ I System/New England Biolabs).Also comprised by this embodiment are functional fragments of any ofthese above recited proteins. The term “functional fragment” as usedherein is intended to denote the capability of said fragment to confersolubility or prevent aggregation.

Further preferred is that the nucleic acid contained in the compositionof the invention is DNA. Particularly preferred is that said DNA iscDNA, synthetic DNA or (semi)synthetic DNA.

Also preferred is that the vector that may be contained in thecomposition of the invention is an expression vector or a gene targetingvector. These vectors may advantageously be used for transfecting hoststhat may or may not be contained in the composition of the invention.

Such vectors may comprise further genes such as marker genes which allowfor the selection of said vector in a suitable host cell and undersuitable conditions. Preferably, the nucleic acid molecule of theinvention is operatively linked to expression control sequences allowingexpression in prokaryotic or eukaryotic cells. Expression of saidnucleic acid molecule comprises transcription of the nucleic acidmolecule into a translatable mRNA. Regulatory elements ensuringexpression in eukaryotic cells, preferably mammalian cells, are wellknown to those skilled in the art. They usually comprise regulatorysequences ensuring initiation of transcription and optionally poly-Asignals ensuring termination of transcription and stabilization of thetranscript. Additional regulatory elements may include transcriptionalas well as translational enhancers, and/or naturally-associated orheterologous promoter regions. Possible regulatory elements permittingexpression in prokaryotic host cells comprise, e.g., the PL, lac, trp ortac promoter in E. coli, and examples for regulatory elements permittingexpression in eukaryotic host cells are the AOX1 or GAL1 promoter inyeast or the CMV-, SV40-, RSV-promoter (Rous sarcoma virus),CMV-enhancer, SV40-enhancer or a globin intron in mammalian and otheranimal cells. Beside elements which are responsible for the initiationof transcription such regulatory elements may also comprisetranscription termination signals, such as the SV40-poly-A site or thetk-poly-A site, downstream of the nucleic acid molecule. Furthermore,depending on the expression system used leader sequences capable ofdirecting the polypeptide to a cellular compartment or secreting it intothe medium may be added to the coding sequence of the nucleic acidmolecule of the invention and are well known in the art. The leadersequence(s) is (are) assembled in appropriate phase with translation,initiation and termination sequences, and preferably, a leader sequencecapable of directing secretion of translated protein, or a portionthereof, into the periplasmic space or extracellular medium. Optionally,the heterologous sequence can encode a fusion protein including an C- orN-terminal identification peptide imparting desired characteristics,e.g., stabilization or simplified purification of expressed recombinantproduct. In this context, suitable expression vectors are known in theart such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia),pCDM8, pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), or pSPORT1 (GIBCO BRL).Preferably, the expression control sequences will be eukaryotic promotersystems in vectors capable of transforming or transfecting eukaryotichost cells, but control sequences for prokaryotic hosts may also beused.

Gene therapy, which is based on introducing therapeutic genes into cellsby ex-vivo or in-vivo techniques is one of the most importantapplications of gene transfer. Suitable vectors and methods for in-vitroor in-vivo gene therapy are described in the literature and are known tothe person skilled in the art; see, e.g., Giordano, Nature Medicine 2(1996), 534–539; Schaper, Circ. Res. 79 (1996), 911–919; Anderson,Science 256 (1992), 808–813; Isner, Lancet 348 (1996), 370–374;Muhlhauser, Circ. Res. 77 (1995), 1077–1086; Wang, Nature Medicine 2(1996), 714–716; WO94/29469; WO 97/00957 or Schaper, Current Opinion inBiotechnology 7 (1996), 635–640, and references cited therein. Thenucleic acid molecules and vectors of the invention may be designed fordirect introduction or for introduction via liposomes, or viral vectors(e.g. adenoviral, retroviral) into the cell. Preferably, said cell is agerm line cell, embryonic cell, or egg cell or derived therefrom, mostpreferably said cell is a stem cell.

Said hosts may then be used for the production of the fusion proteincomprised in the composition of the invention.

Said host cell may be a prokaryotic or eukaryotic cell. The nucleic acidmolecule or vector of the invention which is present in the host cellmay either be integrated into the genome of the host cell or it may bemaintained extrachromosomally.

Preferably, said host is a bacterial, preferably an E. coli, an animal-,preferably a mammalian, an insect-, a plant-, a fungal, preferably ayeast- and most preferably a Saccharomyces or Aspergillius cell, aPichia pastoris cell, a transgenic animal or a transgenic plant.

The present invention also relates to a method of producing a fusionprotein as defined in the diagnostic composition of any of the precedingclaims comprising culturing or raising the host as defined in claim 11and isolating said fusion protein. Preferably the bacterial host E. coliis used for the expression of the GST-HD fusion proteins. The cDNAfragments containing CAG repeats in the normal or pathological rangemay, for example, be cloned into pGEX-5X-1 (Pharmacia) and the resultingplasmids expressing fusion proteins with polygln-sequences used forprotein purification. The resulting proteins may be purified undernative conditions by affinity chromatography on glutathione agarose(Smith and Johnson, 1988).

Additionally, the present invention relates in a preferred embodiment toa composition wherein said antibody is a monoclonal antibody, polyclonalantibody, phage display antibody or a fragment or derivative thereof.

The above recited fragments or derivatives comprise Fv-, Fab-,F(ab)′₂-fragments, single-chain antibodies or single-chain antibodydomains. These antibodies may advantageously be used in experiments suchas Western blotting experiments to determine the presence of the proteinon, for example, nitrocellulose membrane.

Alternatively, the antibody, derivative or fragment thereof may be usedin immunometric assays such as ELISAs or RIAs or may be coupled tocolumns in order to retain the fusion protein from, for example,mammalian sources on said column for further detection or purification.

Alternative uses and advantages of the antibody or fragment orderivative contained in the composition of the invention are, on thebasis of the teachings of this invention, clear to the person skilled inthe art.

The invention further relates to an in vitro method of producing amyloidaggregates comprising (a) at least partially cleaving the fusion proteincomprised in the diagnostic composition of the invention wherein the(poly)peptide that is released has the ability to self-assemble intoamyloid-like fibrils or protein aggregates or (b) inducing self-assemblyinto amyloid-like fibrils or protein aggregates by heating the fusionprotein comprised in the composition of the present invention or anamyloidogenic (poly)peptide that has a ability to self-assemble intoamyloid-like fibrils or protein aggregates by inducing a pH change in asolution comprising said fusion protein/(poly)peptide or by treatingsaid fusion protein/(poly)peptide with a denaturing agent.

The method of the invention may advantageously be used to study in moredetail the process that leads to the formation of amyloid-like fibrilsor protein aggregates from amyloidogenic (poly)peptides. Using themethod of the invention, the onset of, for example, HD or AD can beexamined in an in vitro situation. It is important that the polypeptidethat is released by the cleaving agent still retains the possibility toform amyloid-like fibrils or protein aggregates. The formation of suchfibrils or aggregates may be monitored, for example, by electron orlight microscopy. Varying the cleaving conditions in cases where morethan one cleavage site is present on the fusion protein may be used tofurther elaborate the minimal requirements for said amyloid-like fibrilor protein aggregate formation.

Preferably, the cleavage is effected chemically or enzymatically, or bythe intein self-cleavage reaction in the presence of thiols.

In the following, enzymatic cleavage will also be referred to asproteolytic cleavage. The enzymatic cleavage has the advantage that thecleavage reaction can be performed under almost physiological conditionsand normally only low amounts of the protease are necessary for thecleavage reaction. Furthermore, said cleavage is highly specific and theenzyme can be regarded as nontoxic. Therefore, one can envisage a widevariety if applications within this invention. The disadvantage of theenzymatic cleavage reaction is that prospective inhibitors might inhibitin some cases the protease and in turn prevent the formation of proteinaggregates. In comparison, this is not the case when the cleavagereaction is performed chemically.

Further, the present invention relates to a method of testing aprospective inhibitor of aggregate formation of a fusion protein asdefined in the composition of the invention when enzymatically orchemically cleaved or a non-cleaved fusion amyloidogenic (poly)peptideas defined hereinbefore or an amyloidogenic non-fusion (poly)peptidecomprising

(a) incubating in the presence of a prospective inhibitor

(aa) said fusion protein in the presence or absence of a cleaving agent;or

(ab) said non-fusion poly(peptide); and

(b) assessing the formation of amyloid-like fibrils or proteinaggregates.

This method of the present invention provides a particularly strongimpact on the pharmaceutical research related to amyloid-associateddiseases. For the first time, an inhibitor of fibril or aggregateformation can conveniently, directly, easily and within a short time betested in vitro. As has been detailed herein above, aggregate formationmay be tested on cleavage products, on non-cleaved fusion proteins or onthe above recited non-fusion proteins which have the capacity toaggregate when the temperature is raised, the pH is lowered or theprotein is dissolved in urea and the urea is slowly diluted out with asolvent. Additionally, the present invention does not excludeself-assembly under different conditions.

It was shown recently that acid-mediated denaturation of, e.g.,transthyretin yields a conformational intermediate that canself-assemble into amyloid (Lai et al., 1996). Booth et al. demonstratedthat heat denaturation of human lysozyme variants resulted ininstability, unfolding, and amyloid fibrillogenesis.

Preferably, the incubation is effected in the presence of factor Xa,trypsin, endoproteinase Arg-C, endoproteinase Lys-C, proteinase K,thrombin or elastase at a temperature of preferably 25 to 37° C. for 0,5to 16 hours and the assessment of the formation of fibrils or aggregatesin step (b) is preferably effected by a filter assay or by a thioflavineT (ThT) fluorescence assay, in which the fluorescence intensity reflectsthe degree of aggregation.

As regards the filter assay, a more detailed protocol thereof isexplained in the European patent application entitled “Novel method ofdetecting amyloid-like fibrils or protein aggregates” filed on the sameday with the European Patent Office and assigned to the same applicantwhich is explicitly incorporated herein by reference.

Additionally, the present invention relates to a method for identifyingan inhibitor of aggregate formation of a fusion protein as defined inthe invention prior to or after proteolytic or chemical cleavage or of anon-fusion amyloidogenic (poly)peptide as described herein abovecomprising

-   (a) loading a surface or gel with said protein or an aggregate    thereof;-   (b) incubating said surface or gel with a prospective inhibitor; and-   (c) assessing whether the presence of said prospective inhibitor    avoids or reduces aggregate formation or further aggregate    formation.

In accordance with this embodiment of the invention, proteolytic orchemical cleavage can be advantageously effected either prior or afterthe loading of the surface or gel leaving the investigator additionaldegrees of freedom in devising his experiments. The method of theinvention is both useful for investigating the onset of aggregate orfibril formation or assessing the progression of such a process startingfrom the already existing aggregate or fibril. The latter embodiment isparticularly useful in investigating treatment conditions for patientsthat are already affected by the disease at an early or medium stagethereof.

There is strong evidence that the formation of amyloids is anucleation-dependent polymerization similar to protein crystallizationor microtubule assembly. However, the deposition of a monomer onto apreexisting amyloid template is independent of the nucleation process.Thus, it will be very important to study the deposition of monomers ontoa defined template under physiological conditions. With our in vitrosystem we should be able to monitor the deposition of radiolabelledpolygln-containing monomers.

Preferably, said surface employed in the method of the invention is amembrane. Preferably, the membrane should be cellulose acetate andshould have a low binding capacity for soluble proteins.

The invention also relates to an inhibitor identifiable or identified bythe method of the invention.

The various methods described herein above will give rise to theisolation of a number of inhibitors which are also comprised by thepresent invention. Once such an inhibitor is known, it is of course notnecessary to identify it again by the method of the invention. Rather,said inhibitor can be produced by chemical or recombinant means. In thecase that the inhibitor is of proteinaceous material, it is preferred toresynthesize a compound having the or most of the characteristics ofsaid inhibitor by peptidomimetics.

Preferably, a number of compounds or compound classes are tested fortheir efficacy to inhibit amyloid-like fibril or protein aggregateformation. Said compounds comprise an antibody,4′-Iodo-4′-deoxydoxorubicin (IDOX), pyronine Y, guanidine hydrochloride,urea, rifampicin and derivatives thereof, myristyltrimethylammoniumbromide, hydroquinone, p-benzoquinone, 1,4-dihydroxynaphthalene,p-methoxyphenol, α-tocopherol, ascorbic acid, β-carotene, anthracycline,doxorubicin, hexadecyl-N-methylpiperidinium, dodecyltrimethyl-ammonium,N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, a (poly)peptide,glutamine or an oligoglutamine peptide. These compounds may be used inthe inhibition or reversion of aggregate formation, for example, byformulation into a pharmaceutical composition for the treatment of anyof the diseases cited herein.

The present invention also relates to a pharmaceutical compositioncomprising the inhibitor of the invention and a pharmaceuticallyacceptable carrier and/or diluent.

The pharmaceutical composition of the invention will find wideapplicability in the medical field. Essentially all diseases associatedwith protein aggregate formation or amyloid-like fibril formation, inparticular if they are associated with neuronal tissue or cells, may beeffectively treated with the pharmaceutical composition of theinvention.

Examples of suitable pharmaceutical carriers are well known in the artand include phosphate buffered saline solutions, water, emulsions, suchas oil/water emulsions, various types of wetting agents, sterilesolutions etc. Compositions comprising such carriers can be formulatedby well known conventional methods. These pharmaceutical compositionscan be administered to the subject at a suitable dose. Administration ofthe suitable compositions may be effected by different ways, e.g., byintravenous, intraperitoneal, subcutaneous, intramuscular, topical orintradermal administration. The dosage regimen will be determined by theattending physician and other clinical factors. As is well known in themedical arts, dosages for any one patient depends upon many factors,including the patient's size, body surface area, age, the particularcompound to be administered, sex, time and route of administration,general health, and other drugs being administered concurrently. Atypical dose can be, for example, in the range of 0.001 to 1000 μg (orof nucleic acid for expression or for inhibition of expression in thisrange); however, doses below or above this exemplary range areenvisioned, especially considering the aforementioned factors.Generally, the regimen as a regular administration of the pharmaceuticalcomposition should be in the range of 1 μg to 10 mg units per day. Ifthe regimen is a continuous infusion, it should also be in the range of1 μg to 10 mg units per kilogram of body weight per minute,respectively. Progress can be monitored by periodic assessment. Dosageswill vary but a preferred dosage for intravenous administration of DNAis from approximately 10⁶ to 10¹² copies of the DNA molecule. Thecompositions of the invention may be administered locally orsystemically. Administration will generally be parenterally, e.g.,intravenously; DNA may also be administered directly to the target site,e.g., by biolistic delivery to an internal or external target site or bycatheter to a site in an artery.

The therapeutically useful compounds identified according to the methodof the invention may be administered to a patient by any appropriatemethod for the particular compound, e.g., orally, intravenously,parenterally, transdermally, transmucosally, or by surgery orimplantation (e.g., with the compound being in the form of a solid orsemi-solid biologically compatible and resorbable matrix) at or near thesite where the effect of the compound is desired.

Finally, the present invention relates to a transgenic mammal or plantcomprising a nucleic acid molecule or vector as described in theinvention. The transgenic mammal or plant would advantageously be usedfor the in vivo testing of the efficacy of the inhibitors referred toabove. With the pharmaceutical composition of the invention theformation of protein aggregates in the brain or other tissues of atransgenic animal can be monitored quantitatively. Furthermore, in vivostudies relating to the onset or progress of the above recited diseasesmay be carried out.

DESCRIPTION OF FIGURES AND EMBODIMENTS

FIG. 1

SDS-PAGE Analysis of Purified GST and GST-HD Fusion Proteins.

(a) Aliquots (15 ml) of eluates from the glutathione agarose column weresubjected to 12.5% SDS-PAGE and analyzed by staining with Coomassie blueR. Lanes 1–6 contain GST, GST-HD20, -HD30, -HD83 and -HD122,respectively; lane M contains molecular mass standards. (b) Proteinswere transferred to nitrocellulose and probed with anti-HD1 antibody.Arrows mark the origin of electrophoresis.

FIG. 2

Structure of GST-HD Fusion Proteins

The amino acid sequence (SEQ ID NO:6) corresponding to exon 1 ofhuntingtin is boxed. Arrows labeled Xa and T indicate cleavage sites forfactor Xa and trypsin, respectively.

FIG. 3

Site-Specific Proteolysis of GST-HD Fusion Proteins with Trypsin andFactor Xa.

Tryptic digestions were performed at 37° C. for 3 (a) or 16 h (b).Native proteins and their cleavage products were subjected to 12.5%SDS-PAGE, blotted onto nitrocellulose membranes, and probed withanti-HD1 antibody. Arrows mark the origin of electrophoresis. (c)Purified fusion proteins and their factor Xa and trypsin cleavageproducts were analyzed using the filter retardation assay. The proteinsretained by the cellulose acetate and nitrocellulose membranes weredetected by incubation with the anti-HD1 antibody.

FIG. 4

Electron Micrographs of Native GST-HD Fusion Proteins and their FactorXa and Trypsin Cleavage Products.

Purified GST fusion proteins were protease treated, negatively stainedwith uranyl acetate and viewed by electron microscopy. The undigestedGST-HD51 molecules appear as a homogeneous population of small, roundparticles (a).

Removal of the GST-tag with factor Xa results in the formation ofamyloid-like fibrils and intermediate structures (b+c). After partialdigestion (3 h) of GST-HD51 with trypsin, the ribbons are associatedwith terminal clots (d, arrow), whereas prolonged digestion (16 h)produces ribbons without attached clots (e). Removal of the GST-tag fromGST-HD20 shows no evidence for the formation of defined structures (f).

FIG. 5

Birefringence of Protein Aggregates Formed by Proteolytic Cleavage ofGST-HD51.

The protein aggregates were stained with Congo red. (a) Bright field,200×; (b) Polarized light, 200X; (c) Polarized light, 100X.

FIG. 6

Polygln-Containing Protein Aggregates are Formed in vivo.

(a) Western blot analysis, after separation by 10% SDS-PAGE, of thenuclear (N) and cytosolic (C) protein fractions prepared from brain andkidney of an R6/2 hemizygous transgenic mouse and a littermate control.Blots were probed with anti-HD1, anti-GAPDH and anti-Fos B antibodies asindicated. (b) Detection of HD exon 1 protein aggregates formed in vivousing the cellulose acetate filter assay. The membrane was immunostainedusing the anti-HD1 antibody. (c) Ultrastructure of a neuronalintranuclear inclusion (NII). The presence of a NII in a striatal neuronof a 17 month old R6/5 homozygous mouse is shown. The NII is indicatedby the large arrow and the fibrillar amyloid-like structures within theNII are indicated by two small arrows. The scale bar is 250 nm.

FIG. 7

Proposed Mechanism for the Formation of Amyloid-like Fibrils byProteolytic Cleavage of GST-HD51

GST-HD51 molecules are represented as follows: zigzag line, elongatedpolygln repeat forming a stable hairpin with β-sheet structure; dottedline, N-terminal amino acids in the HD exon 1 protein containing thefactor Xa cleavage site (arrow) (undefined structure); thin line,C-terminal amino acids in the HD exon 1 protein (undefined structure);shaded symbol, the dimeric globule-like form of GST.

Prior to cleavage, the HD exon 1 protein is tightly bound to the GST-tagpreventing intermolecular interactions (1). Removal of the GST-tag withfactor Xa renders the polygln repeat accessible allowing the formationof fibrils as seen by EM (3). During cleavage, intermediate structuresform through specific polygln interactions before complete release ofthe GST-tag has occurred (2). These intermediates appear as clots underEM and are frequently seen at the terminals of growing fibrils.

FIG. 8

Structure of GST-HD fusion proteins (SEQ ID NOS:38–41, respectively inorder of appearance). The amino acid sequences corresponding to theN-terminal portion of huntingtin are boxed and the amino acidscorresponding to the biotinylation site are underlined. Arrows labeled(Xa) and (T) indicate cleavage site for factor Xa and trypsin,respectively.

FIG. 9

Detection of polyglutamine-containing protein aggregates formed in vitroand in transfected COS-1 cells using the dot-blot filter retardationassay. (A) Purified GST-HD20DP and -HD51 DP fusion proteins (250 ng) andtheir factor Xa and trypsin cleavage products were applied to the filteras indicated. The aggregated proteins retained by the cellulose acetatemembrane were detected by incubation with the anti-HD1 antibody. (B)Scanning electron micrograph of aggregated GST-HD51DP trypsin cleavageproducts retained on the surface of the cellulose acetate membrane(Photo: Heinrich Lündsdorf, GBF Braunschweig, Germany). (C) Dot-blotfilter retardation assay performed on the insoluble fraction isolatedfrom transfected and non-transfected COS-1 cells. COS-1 cells weretransiently transfected with the plasmids pTL1-CAG20, -CAG51 and CAG93encoding huntingtin exon 1 proteins with 20 (HD20), 51 (HD51) and 93(HD93) glutamines, respectively. The pellet fractions obtained aftercentrifugation of whole cell lysates were subjected to DNasel/trypsindigestion, boiled in 2% SDS, and portions of 1, 3 and 6 μl were filteredthrough a cellulose acetate membrane. The aggregated huntingtin proteinretained on the membrane was detected with the anti-HD1 antibody. NT,non-transfected cells.

FIG. 10

Detection and quantification of aggregates formed in vitro frombiotinylated GST-HD exon 1 fusion proteins. Various amounts of thefusion proteins GST-HD51DPBio and -HD20DPBio were filtered through acellulose acetate membrane after a 3-h incubation at 37° C. in thepresence or absence of trypsin as indicated. (10A) Images of theretained protein aggregates, detected with streptavidin-AP conjugateusing either a fluorescent (upper panel) or a chemiluminescent APsubstrate (lower panel). (10B) Quantification of signal intensitiesobtained for the GST-HD51DPBio dots seen in A. Fluorescence andchemiluminescence values are arbitrary units generated by theLumi-Imager F1 and LumiAnalyst™ software (Boehringer Mannheim).

FIG. 11

Detection (11A) and quantification (11B) of aggregates formed in vitrofrom biotinylated GST-HD exon 1 fusion proteins using the dot-blot andmicrotitre plate filter retardation assay. Various amounts of the fusionproteins GST-HD51DPBio and -HD20DPBio were filtered through thecellulose acetate membranes after a 3-h incubation at 37° C. in thepresence or absence of trypsin as indicated. The detection andquantification of the aggregates was as described in FIG. 3.

FIG. 12

Detection of neurofibrillar tangles (NFTS) and β-amyloids in brainextracts prepared from Alzheimer's disease patients and controls usingthe dot-blot filter retardation assay. The cellulose acetate membranewas probed with the polyclonal anti-Tau, the monoclonal anti-β-amyloid,or the polyclonal anti-HD antibody. A1, A2, and A3: protein extractsprepared from cerebral cortex of Alzheimer's disease patients; C1, C2,and C3: protein extracts prepared from cerebral cortex of normalindividuals. GST-HD51, fusion of glutathione S-transferase andhuntingtin exon 1 containing 51 glutamines.

The examples illustrate the invention:

EXAMPLE 1

Purification of GST-HD Fusion Proteins Containing Expanded Polyglns

Exon 1 of the HD gene was isolated from genomic phage clones, derivedfrom the normal and expanded alleles of an HD patient (Sathasivam etal., 1997), and used for the expression of GST-HD fusion proteins in E.coli. DNA fragments containing CAG repeats in the normal (CAG)₂₀₋₃₃ andexpanded (CAG)₃₇₋₁₃₀ range were cloned into pGEX-5X-1 (Pharmacia), andthe resulting plasmids expressing fusion proteins with 20 (GST-HD20), 30(-HD30), 51 (-HD51), 83 (-HD83) and 122 (-HD122) glutamines,respectively, were used for protein purification. For plasmidconstruction lambda phage from stock 9197₄ (Sathasivam et al., 1997)were plated to give single plaques which were inoculated into 400 mlcultures of E. coli XL1-Blue MRF′ (Stratagene) for DNA preparation. TheDNA sequence encoding the N-terminal portion of huntingtin (exon 1),including the CAG repeats, was amplified by PCR using the following pairof primers: ES 25 (TGGGATCCGCATGGCGACCCTGGAAAAGCTGATGAAGG) (SEQ IDNO: 1) corresponding to nt315-343 of the HD gene (HDCRG, 1993) andcontaining a BamHI site (underlined) and ES 26(GGAGTCGACTCACGGTCGGTGCAGC GCTCCTCAGC) (SEQ ID NO: 2) corresponding tont516-588 and containing a Sail site (underlined). Conditions for PCRwere as described (Mangiarini et al., 1996). Due to instability of theCAG repeat during propagation in E. coli, DNA preparations fromindividual plaques yielded different sized PCR products. Fragments of˜320, 360, 480 and 590 bp were gel-purified digested with BamHI and SaIIand inserted into the BamHI-SaII site of the expression vector pGEX-5X-1(Pharmacia), yielding pCAG30, pCAG51, pCAG83 and pCAG122, respectively,pCAG20, containing 20 repeats of CAG within the cloned HD exon 1sequence, was similarly constructed from a phage genomic clone derivedfrom a normal allele. All constructs were verified by sequencing. Afterinduction with IPTG, the resulting proteins were purified under nativeconditions by affinity chromatography on glutathione agarose. Thus, E.coli SCS1 (Stratagene) carrying the pGEX expression plasmid of interestwas grown to an OD_(600nm) of 0.6 and induced with IPTG (1 mM) for 3.5 has described in the manufacturer's protocol (Pharmacia). Cultures (200ml) of induced bacteria were centrifuged at 4000 g for 20 min, and theresulting pellets were stored at −80° C. Cells were thawed on ice andresuspended in 5 ml of lysis buffer (50 mM sodium phosphate, 150 mMNaCl, 1 mM EDTA, pH 7.4) containing 0.5 mg/ml lysozyme. After 45 min at0° C., cells were sonicated with two 30 sec-bursts.Octyl-β-D-glucopyranoside was then added to a final concentration of0.1% and the resulting lysate was clarified by centrifugation at 30,000g for 30 min at 4° C. Cleared lysates were incubated for 1 h at 4° C.with 500 μl of a 1:1 slurry of glutathione-agarose beads (Sigma) thathad been washed times and resuspended in lysis buffer. The beads werepoured into a small column and washed extensively with lysis buffercontaining 0.1% octyl-β-D0glucopyranoside. The bound fusion protein waseluted with 2 ml of 15 mM glutathione (reduced) in lysis buffer. Typicalyields were 0.5–1 mg of purified GST-HD20, -HD30 and -HD51 proteins per200 ml of bacterial culture; yields of GST-HD83 and -HD122 were muchlower, less than 10% of that obtained with the shorter fusion proteins.Protein was determined by the Bio-Rad dye binding assay using bovineserum albumin as standard. SDS-PAGE of the purified GST-HD20, -HD30,-HD51, -HD83 and -HD122 proteins revealed major bands of 42, 45, 50, 65and 75 kDa, respectively (FIG. 1 a). These bands were also detected whenthe various protein fractions were subjected to immunoblot analysisusing the affinity purified anti-huntingtin antibody HD1 (FIG. 1 b,lanes 2–6). HD1 specifically detects the GST-HD fusion proteins onimmunoblots, whereas the GST-tag alone is not recognized (FIG. 1 b, lane1). For immunoblotting a bacterial plasmid encoding HD1-His, aHis₆-tagged fusion protein containing residues 1–222 of huntingtin, wasgenerated by inserting a PCR-amplified IT-15 cDNA fragment into thepQE-32 vector (Qiagen). The fusion protein was expressed in E. coli,affinity-purified under denaturing conditions on Ni-NTA agarose, andinjected into rabbits. The resulting immune serum was thenaffinity-purified against the antigen that had been immobilized onNi-NTA agarose. The GAPDH- and Fos B-specific antisera have beendescribed (Wanker et al., 1997; Davies et al., 1997).

Western blotting was performed as detailed (Towbin et al., 1979). Theblots were incubated with 1:1000 dilutions of the indicated primaryantibody, followed by an alkaline-phosphatase-conjugated secondaryantibody. Color development was carried out with5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium assubstrates (Promega).

All recombinant proteins migrated at a size corresponding nearly to thatpredicted from their amino acid sequence. Interestingly, an additionalhigh molecular weight band which remains at the top of the gel, wasconsistently detected in the protein fractions with the longest polyglns(83 and 122 residues; FIGS. 1 a and b, lane 5 and 6). This band was mostprominent on the immunoblots but was also clearly detectable in theCommassie stained gel. This immunoreactive material was often stillpresent at the bottom of the loading slots, even after the samples hadbeen boiled for 5 min in the presence of 2% SDS and 6 M urea prior toloading.

EXAMPLE 2

Proteolytic Cleavage of GST-HD Fusion Proteins Containing ExpandedPolyglns

It has been shown previously that the solubility of certain proteins canbe enhanced by the addition of the GST-tag (Smith and Johnson, 1988) andit was therefore of interest to determine whether the removal of theGST-tag by proteolytic cleavage would have an effect on the solubilityof the polygln-containing fusion proteins. Potential factor Xa andtrypsin cleavage sites within the GST-HD fusion proteins are shown inFIG. 2. Factor Xa cleaves between the GST-tag and the HD exon 1 proteinwhereas trypsin removes an additional 15 amino acids from the N-terminusand a single proline from the C-terminus, both proteases leaving thepolygln repeat intact. The GST-HD20, -HD30 and -HD51 proteins weredigested with trypsin under conditions designed to remove the GST-tagfrom the fusion protein without it being totally degraded. Aftercleavage, proteins were denatured by boiling in the presence of 2% SDSand analyzed by SDS-PAGE and immunoblotting using the anti-HD1 antibody.GST-HD20 and -HD30 cleavage yielded products migrating in a 12:5% gel atapproximately 30 and 33 kDa, respectively. In contrast, cleavage ofGST-HD51 resulted in the formation of two protein products migrating atapproximately 37 and 60 kDa, and an additional weak immunoreactive bandon the bottom of the loading slots was also detected (FIG. 3 a). Thishigh molecular weight band was more pronounced when GST-HD51 wasdigested with trypsin under conditions in which the GST-tag was totallydegraded (FIG. 3 b). However, with proteins GST-HD20 and -HD30 thislonger exposure to trypsin produced the same cleavage products as theones seen in FIG. 3 a and the high molecular weight products were notobserved. Similar results were obtained with factor Xa protease andendoproteinases Arg-C and Lys-C. As regards the proteolytic cleavages,the following protocols were carried out: The GST-HD fusion proteinspurified as described above were dialysed against 40 mM Tris-HCl (pH8.0), 150 mM NaCl, 0.1 mM EDTA and 5% (v/v) glycerol to raise the pHprior to proteolytic cleavage. The proteins were then combined withbovine factor Xa (New England Biolabs) or modified trypsin (BoehringerMannheim, sequencing grade) in dialysis buffer containing 2 mM CaCl₂ atan enzyme:substrate ratio of 1:10 (w/w) or 1:40 (w/w), respectively.Incubations with factor Xa were at 25° C. for 16 h. Tryptic digestionswere performed at 37° C. for 3 or 16 h as indicated. Digestions wereterminated by the addition of PMSF to 1 mM. The degree of proteolysiswas determined by SDS-PAGE followed by staining with Coomassie blue orimmunoblottting using anti-HD1 antibody.

We have developed a simple and sensitive filter assay to detect theformation of high molecular weight insoluble protein aggregates. Thisassay is based on the finding that the SDS-insoluble protein aggregatesobtained by proteolytic cleavage of GST-HD51 are retained on a celluloseacetate filter, whereas the soluble cleavage products of GST-HD20 andGST-HD30 are not. Factor Xa or trypsin digestions of purified GST-HDfusion proteins (10 μg) were performed in a 20 μl reaction mixture asdescribed above. Reactions were terminated by adjusting the mixture to2% SDS and 50 mM DTT. After heating at 100° C. for 5 min, aliqouts (0.5μl) were diluted into 200 μl of 0.1% SDS and filtered through acellulose acetate membrane (Schleicher & Schuell, 0.2 μm pore size)using a BRL dot blot filtration unit. Filters were washed with water,and the SDS-insoluble aggregates retained on the filter detected byincubation with the anti-HD1 antibody, followed by an anti-rabbitsecondary antibody conjugated to alkaline phosphatase (BoehringerMannheim). FIG. 3 c shows immunoblots of cellulose acetate andnitrocellulose membranes to which the native GST-HD20, -HD30 and -HD51proteins and their factor Xa and trypsin cleavage products have beenapplied. On the cellulose acetate filter, only the cleavage products ofGST-HD51 were detected by the anti-HD1 antibody, indicating theformation of insoluble high molecular weight protein aggregates. Incontrast, all the uncleaved GST-HD fusion proteins and their digestionproducts were detected on the nitrocellulose control filter. This assaywas also used to detect huntingtin aggregates present in a nuclearfraction from the brain of an R6/2 hemizygous mouse and littermatecontrol (see preparation of nuclei below).

EXAMPLE 3

Huntingtin Proteins Containing Expanded Polyglns in the PathologicalRange Aggregate to Amyloid-Like Birefringent Fibrils

Electron microscopy of negatively stained GST-HD51 fractions showedoligomeric particles with diameters of 6 to 7 nm (FIG. 4 a); no higherordered aggregates were observed. For electron microscopic observation,the native or protease-digested GST-HD fusion proteins were adjusted toa final concentration of 50 μg/ml in 40 mM Tris-HCl (pH 8.0), 150 mMNaCl, 0.1 mM EDTA and 5% glycerol. Samples were negatively stained with1% uranyl acetate and viewed in a Philips CM100 EM. In contrast, proteinfractions obtained by proteolytic cleavage of GST-HD51 showed numerousclusters of high molecular weight fibrils and ribbon-like structures(FIGS. 4 b, c, d and e), reminiscent of purified amyloids (Prusiner etal., 1983). The fibrils obtained after digestion with factor Xa showed adiameter of 10–12 nm and their length varied from 100 nm up to severalmicrometers (FIGS. 4 b and c). In the trypsin-treated samplesribbon-like structures formed by lateral aggregation of fibrils with adiameter of 7.7 nm were observed (FIGS. 4 d and e). After treatment withfactor Xa or limited digestion with trypsin, clots of small particleswere frequently detected on one or both ends of the fibrils (FIGS. 4 b,c and d). These clots of varying sizes and shapes were not seen whenGST-HD51 was digested with trypsin under conditions in which the GST-tagis totally degraded (FIG. 4 e), indicating that they contain GST. Instrong contrast to GST-HD51, the GST-HD20 and -HD30 proteins did notshow any tendency to form ordered high molecular weight structures,either with or without protease treatment (FIG. 4 f).

The insoluble protein aggregates formed by proteolytic cleavage ofGST-HD51 were isolated by centrifugation and stained with Congo red(Caputo et al., 1992) and examined under a light microscope. For lightmicroscopy, peptide aggregates formed by trypsin digestion of purifiedGST-HD fusion proteins (50 μg in 100 μl of digestion buffer) werecollected by centrifugation at 30,000 g for 1 h and resuspended in 10 μlof water. Samples were mixed with 0.1 volume of a 2% (w/v) aqueous CongoRed (Sigma) solution, placed on aminoalkylsilane-coated glass slides,and allowed to dry overnight under a coverslip. After removing thecoverslip, excess Congo Red was removed by washing with 90% ethanol.Evaluation of the Congo Red staining by polarization microscopy wasperformed using a Zeiss Axiolab Pol microscope equipped with strain-freelenses and optimally aligned cross-polarizers. After staining, theprotein aggregates on the glass slides were red, indicating that theyhad bound the dye (FIG. 5 a), and when examined under polarized light agreen color and birefringence were detected (FIGS. 5 b and c). Thesestaining characteristics were similar to those observed for prions(Prusiner et al., 1983) and amyloids (Caputo et al., 1992).

EXAMPLE 4

Huntingtin Proteins Containing Expanded Polyglns Form Amyloid-LikeProtein Aggregates In Vivo

To determine whether the amyloid-like protein aggregates formed byproteolytic cleavage of GST-HD51 in vitro are also present in vivo,nuclear protein fractions of brain and kidney were prepared from micetransgenic for the HD mutation (line R6/2) and littermate controls(Davies et al., 1997; Mangiarini et al., 1996). Nuclei from the brain orkidney of an R6/2 hemizygous mouse with a repeat expansion of (CAG)₁₄₃(Mangiarini et al., 1996) at ten weeks of age and littermate controlwere prepared as follows. Whole brain samples (80 mg) in 400 ml of 0.25M sucrose in buffer A (50 mM triethanolamine [pH 7.5], 25 mM KCl, 5 mMMgCl₂, 0.5 mM DTT, 0.5 mM PMSF) were homogenized using 15 strokes of atight-fitting glass homogenizer. The homogenate was adjusted to a finalconcentration of 5 mM DTT, and centrifuged at 800 g for 15 min. Thesupernatant was recentrifuged at 100,000 g for 1 h, and the supernatantfrom this centrifugation was taken as the cytosolic fraction (fractionC). The loose pellet from the first centrifugation was homogenized,diluted to 1.2 ml with 0.25 M sucrose/buffer A, and mixed with twovolumes of 2.3 M sucrose/buffer A. The mixture was then layered on topof 0.6 ml 2.3 M sucrose/bufferA in a SW60 tube and centrifuged at124,000 g for 1 h. The pellet was harvested with a spatula, resuspendedin 200 μl of 0.25 M sucrose/buffer A and again centrifuged at 800 g for15 min. The entire procedure was carried out at 4° C. The pelletednuclei were resuspended to a density of ˜1×10⁷ nuclei/ml in 0.25sucrose/buffer A (fraction N) and stored at −80° C. Nuclei from mousekidney were prepared in the same way. The protein extracts were analyzedby SDS-PAGE and Western blotting using the anti-HD1 antibody (FIG. 6 a).Strikingly, this antibody detected a prominent high molecular weightband in the nuclear fraction (N) prepared from R6/2 transgenic brain,very similar to the high molecular weight band obtained by proteolyticcleavage of GST-HD51 (FIG. 3 b). No such immunoreactive band wasdetected in the nuclear fraction of brain from the littermate controland it was also absent from the corresponding cytoplasmic fractions (C).A small amount of high molecular weight material was also detected inthe nuclear fraction prepared from R6/2 transgenic kidney, but was againabsent from the cytoplasmic fraction. The purity of the nuclear andcytoplasmic fractions was confirmed by Western blot analysis using theanti-Fos B and anti-GAPDH antibodies. Anti-Fos B detected thetranscription factor mainly in the nuclear fraction, and the enzymeGAPDH was only seen in the cytoplasmic fraction, as expected. TheWestern blot results were reproduced using the cellulose acetate filterassay (FIG. 6 b). Using this assay, a 10–20 fold higher amount oftransgene protein was detected in the nuclear fraction isolated frombrain material, compared to that prepared from kidney.

The formation of NIIs has been shown to preceed the neuronal dysfunctionthat forms the basis of the progressive neurological phenotype observedin the R6 transgenic lines (Davies et al., 1997). These NIIs areimmunoreactive for both huntingtin and ubiquitin antibodies and containthe transgene but not the endogenous huntingtin protein. Therefore,Western blot analysis using an anti-ubiquitin antibody was alsoperformed showing the same pattern of immunoreactivity as had beenobserved with the anti-HD1 antibody (FIG. 6 a), and indicating that thehigh molecular weight transgene protein present in the nuclear fractionis ubiquitinated (data not shown).

To examine whether the NIIs containing the proteins huntingtin andubiquitin (Davies et al., 1997) have a fibrous composition, anultrastructural analysis was performed. Experimentally, a 17 month oldR6/5 homozygous mouse ((CAG)₁₂₈₋₁₅₅) (Mangiarini et al., 1996) wasdeeply anaesthetised with sodium pentobarbitone and then perfusedthrough the left cardiac ventricle with 35–50 ml of 4% paraformaldehydeand either 0.5% glutaraldehyde in 0.1 M Millonig's phosphate buffer (pH7.4). The brain was removed from the skull and placed in fresh fixativeovernight at 4° C. Coronal sections (50–200 μm) were cut on an OxfordVibratome (Lancer) and collected in serial order in 0.1 M phosphatebuffer. After being osmicated (30 min in 1% OsO₄ in 0.1 M phosphatebuffer) the sections were stained for 15 min in 0.1% uranyl acetate insodium acetate buffer at 4° C., dehydrate in ethanols, cleared inpropylene oxide and embedded in Araldite between two sheets of Melanex(ICI). Semi thin (1 μm) sections were cut with glass knife on a ReichertUltracut ultramicrotome. The sections were collected on mesh gridscoated with a thin formvar film, counterstained with lead citrate andviewed in a Jeol 1010 electron microscope. An electron micrograph of aNII from a 17 month old R6/5 homozygous mouse is shown in FIG. 6 c. ThisNII (large arrow) contains high molecular weight fibrous structureswhich were clearly differentiated from the surrounding chromatin. Thefilaments were randomly oriented, 5–10 nm in diameter and often measuredup to 250 nm in length (small arrows). These structures differ fromthose previously reported in the NIIs seen in hemizygous R6/2 mice whichwere far more granular in composition, with individual filamentousstructures being more difficult to distinguish (Davies et al., 1997).R6/2 mice exhibit an earlier age of onset with a more rapid progressionof the phenotype and do not survive beyond 13 weeks (Mangiarini et al.,1996). It is possible that the filamentous structures do not have timeto form in the R6/2 mice.

EXAMPLE 5

Construction of Further Plasmids, Purification of Corresponding GSTFusion Proteins and Proleolytic Cleavage of GST Fusion Proteins

In a second set of experiments, a further number of plasmids wasconstructed. Standard protocols for DNA manipulations were followed (J.Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: ALaboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y., 1989). IT-15 cDNA sequences (HDCRG, Cell 72, 971(1993)) encoding the N-terminal portion of huntingtin, including the CAGrepeats, were amplified by PCR using the oligonucleotides ES25(5′-TGGGATCCGCATGGCGACCCTGGAAAAGCTGATGA AGG-3′) (SEQ ID NO: 1) and ES27(3′-CTCCTCGAGCGGCGGTGGCGGC TGTTGCTGCTGCTGCTG-5′) (SEQ ID NO: 3) asprimers and the plasmids pCAG20 and pCAG51 as template (E. Scherzinger,R. Lurz, M. Trumaine, L. Margiarini, B. Hollenbach, R. Hasenbank, G. P.Bates, S. W. Davies, H. Lehrach, and E. E. Wanker, Cell 90, 549 (1997)).Conditions for PCR were as described (L. Mangiarini, K. Sathasivam, M.Seller, B. Cozens, A Harper, C. Hetherington, M. Lawton, Y Trottier, H.Lehrach, S. W. Davies, and G. P. Gates, Cell 87, 493 (1996)). Theresulting cDNA fragments were gel purified, digested with Bam HI and XhoI and were inserted into the Bam HI-Xho I site of the expression vectorpGEX-5X-1 (Pharmacia), yielding pCAG20DP and pCAG51DP, respectively. Theplasmids pCAG20DP-Bio and pCAG51DP-Bio were generated by subcloning thePCR fragments obtained from the plasmids pCAG20 and pCAG51 intopGEX-5X-1-Bio. PGEX-5X-1-Bio was created by ligation of theoligonucleotides BIO1 (5′-CGCTCGAGGGTATCTTCGAGGCCCAGAAGATCGAGTGGCGATCACCATGAG-3′) (SEQ ID NO: 4) and BIO2 (5′-GGCCGCTCATGGTGATCGCCACTCGATCTTCTGGGCCTCGAAGATACCCTCGAG-3′) (SEQ ID NO: 5), afterannealing and digestion with Xho I, into the Xho I-Not I site ofpGEX-5X-1. The plasmids with the IT-15 cDNA inserts were sequenced toconfirm that no errors had been introduced by PCR. The construction ofplasmids pTL1-CAG20, pTL1-CAG51 and pTL1-CAG93 for the expression ofhuntingtin exon 1 proteins containing 20, 51 and 93 glutamines inmammalian cells has been described (A. Sittler, S. Walter, N. Wedemeyer,R. Hasenbank, E. Scherzinger, G. P. Bates, H. Lehrach, and E. E. Wanker,Mol. Cell, submitted).

The amino acid sequence of the GST-HD fusion proteins encoded by the E.coli expression plasmids pCAG20DP, pCAG51DP, pCAG20Dp-Bio andpCAG51DP-Bio is shown in FIG. 8. The plasmids pCAG20DP and pCAG51DPencode fusion proteins of glutathione S-transferase (GST) and theN-terminal portion of huntingtin containing 20 (GST-HD20DP) and 51(-HD51DP) polyglutamines, respectively. In these proteins theproline-rich region located immediately downstream of the glutaminerepeat was deleted (E. Scherzinger, R. Lurz, M. Trumaine, L. Margiarini,B. Hollenbach, R. Hasenbank, G. P. Bates, S. W. Davies, H. Lehrach, andE. E. Wanker, Cell 90, 549 (1997). The fusion proteins GST-HD20DPBio and-HD51DPBio are identical to GST-HD20DP and -HD51DP, except for thepresence of a biotinylation site (P. J. Schatz, Biotechnology 11, 1138(1993)) at their C-termini.

In the experiments described herein, E. coli DH10B (BRL) was used forplasmid construction and E. coli SCS1 (Stratagene) was used for theexpression of GST-HD fusion proteins. Transformation of E. coli withplasmids and ligation mixtures was performed by electroporation using aBio-Rad Gene Pulser (Richmond, Calif.). Transformed cells were spread onLB plates supplemented with appropriate antibiotics (J. Sambrook, E. F.Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory Press, Plainview, N.Y. 1989). Forexpression of GST fusion proteins, cells were grown in liquid TY medium(5 g NaCl, 5 g yeast extract, and 10 g tryptone per liter) buffered with20 mM MOPS/KOH (pH 7.9) and supplemented with glucose (0.2%), thiamine(20 μg/ml), ampicillin (100 μg/ml) and kanamycin (25 μg/ml).

The procedure for purification of GST fusion proteins is an adaption ofthe protocol of Smith and Johnson (D. B. Smith and K. S. Johnson, Gene67, 31 (1988)). Unless indicated otherwise, all steps were performed at0–4° C.

First, 100 ml TY medium were inoculated with a single colony containingthe expression plasmid of interest, and the culture was incubated at 37°C. overnight with shaking. Then, 1.5 liter TY medium were inoculatedwith the overnight culture and grown at 37° C. until an OD₆₀₀ of 0.6 wasreached. IPTG was added to a final concentration of 1 mM, and theculture continued to grow at 37° C. for 3.5 h with vigorous shaking. Theculture was chilled on ice, and the cells harvested by centrifugation at4000× g for 20 min.Cells were washed with buffer A [50 mM sodium phosphate (pH 8), 150 mMNaCl, and 1 mM EDTA]. If neccessary, the cell pellet was stored at −70°C. Cells were resuspended in 25 ml buffer A. PMSF and lysozyme(Boehringer Mannheim) were added to 1 mM and 0.5 mg/ml, respectively,and incubated on ice for 45 min. Cells were lysed by sonication (2×45 s,1 min cooling, 200–300 Watt), and Triton X-100 was added to a finalconcentration of 0.1% (v/v). The lysate was centrifuged at 30.000× g for30 min, and the supernatant was collected.5 ml of a 1:1 slurry of GST-agarose (Sigma), previously equilibrated inbuffer A, was added and the mixture was stirred for 30 min. The slurrywas poured into a 1.6 cm diameter column, washed once with 40 ml bufferA containing 1 mM PMSF and 0.1% Triton X-100 and twice with 40 ml bufferA containing 1 mM PMSF. The protein was eluted with 5×2 ml buffer Acontaining 15 mM reduced glutathione (Sigma). Aliquots of the fractionswere analyzed by SDS-PAGE and the fractions containing purified GSTfusion protein were combined. Finally, the pooled fractions weredialysed overnight against buffer B [20 mM Tris/HCl (pH 8), 150 mM NaCl,0.1 mM EDTA and 5% (v/v) glycerol], aliquotted, freezed in liquidnitrogen and stored at −70° C.Typical yields were 10–20 mg for GST-HD20DP and -HD51DP and 5–10 mg forGST-HD20DPBio and -HD51DPBio per liter of bacterial culture. Proteinconcentration was determined using the Coomassie protein assay reagentfrom Pierce with BSA as a standard.The GST-huntingtin fusion proteins (2 mg) were digested with bovinefactor Xa (New England Biolabs) or with modified trypsin (BoehringerMannheim, sequencing grade) at an enzyme/substrate ratio of 1:10 (w/w)and 1:20 (w/w), respectively. The reaction was carried out in 20 μl of20 mM Tris/HCl (pH 8), 150 mM NaCl and 2 mM CaCl₂. Incubations withfactor Xa were performed at 25° C. for 16 h. Tryptic digestions were at37° C. for 3 to 16 h. Digestions were terminated by the addition of 20μl 4% (w/v) SDS and 100 mM DTT, followed by heating at 98° C. for 5 min.As shown in the previous examples, removal of the GST tag from the HDexon 1 protein containing 51 glutamines (GST-HD51) by site-specificproteolytic cleavage results in the formation of high molecular weightprotein aggregates, seen as characteristic fibrils or filaments onelectron microscopic examination. Such ordered fibrillar structures werenot detected after proteolysis of fusion proteins containing only 20(GST-HD20) or 30 (GST-HD30) glutamines, although light scatteringmeasurements (Y. Georgalis, E. B. Starikov, B. Hollenbach, R. Lurz, E.Scherzinger, W. Saenger, H. Lehrach, and E. E. Wanker, Proc. Natl. Acad.Sci. USA 95, 6118 (1998)) revealed that some form of aggregation alsooccured with these normal repeat-length proteins. In the presentexample, truncated GST-HD exon 1 fusion proteins with or without aC-terminal biotinylation tag (P. J. Schatz, Biotechnology 11, 1138(1993) were used. These fusion proteins contain either 20 or 51glutamines but lack most of the proline rich region located downstreamof the glutamine repeat (E. Scherzinger, R. Lurz, M. Trumaine, L.Margiarini, B. Hollenbach, R. Hasenbank, G. P. Bates, S. W. Davies, H.Lehrach, and E. E. Wanker, Cell 90, 549 (1997)). Potential factor Xa andtrypsin cleavage sites within the GST-HD fusion proteins are shown inFIG. 8. As outlined above, the proteins GST-HD20DP and -HD51DP wereexpressed in E. coli and affinity-purified under native conditions. Theywere then digested overnight with trypsin or faxtor Xa protease topromote the formation of polyglutamine-containing huntingtin aggregates.FIG. 9A shows an immunoblot of a cellulose acetate membrane to which thenative GST-HD20DP and -HD51DP proteins and their factor Xa and trypsincleavage products have been applied.To monitor the in vitro formation of polyglutamine-containing aggregateswithout the need for a specific antibody, a modified filter retardationassay was developed. In this assay, streptavidin-conjugated alkalinephosphatase (AP) is used to detect the insoluble protein aggregatesretained on the cellulose acetate filter membrane. Streptavidin bindsspecifically to the biotinylation tag (P. J. Schatz, Biotechnology 11,1138 (1993)) that has been added C-terminal to the polyglutamine tractin the fusion proteins GST-HD20DPBio and -HD51DPBio (FIG. 7) (seeExample 8 for details). FIG. 10A shows that the modified aggregationassay gives results comparable to those obtained with thenon-biotinylated fusion proteins in that insoluble aggregates areproduced from the trypsin-treated GST-HD51DPBio protein but not from theuncleaved GST-HD51DPBio protein or the corresponding 20 repeat samples.Using either fluorescent (AttoPhos™) or chemiluminescent (CDP-Star™)substrates for alkaline phosphatase, it is possible to capture andquantify the filter assay results with the Boehringer Lumi-Imager F1system. With both AP substrates, aggregates formed from as little as5–10 ng of input GST-HD51DPBio protein were readily detected on thecellulose acetate membrane, and signal intensities increased linearly upto 250 ng of fusion protein applied to the filter (FIG. 10B).

EXAMPLE 6

Isolation of Amyloid-Like Protein Aggregates from Transfected COS-1Cells

To examine whether polyglutamine-containing aggregates are also formedin vivo, HD exon 1 proteins with 20, 51 or 93 glutamines (without a GSTtag) were expressed in COS-1 cells. Whole cell lysates were prepared,and after centrifugation, the insoluble material was collected andtreated with DNasel and trypsin to lower the viscosity. The resultingprotein mixture was then boiled in SDS and analyzed using the dot-blotfilter retardation assay (see Example 8). In more detail, the followingexperimental protocol was carried out:

COS-1 cells were grown in Dulbecco's modified Eagle medium (Gibco BRL)supplemented with 5% (w/v) fetal calf serum (FCS) containing penicillin(5 U/ml) and streptomycin (5 μg/ml), and transfection was performed asdescribed (A. Sittler, D. Devys, C. Weber, and J.-L. Mandel, Hum. Mol.Genet. 5, 95 (1996)).COS-1 cells transfected with the mammalian expression plasmidspTL1-CAG20, pTL1-CAG51 and pTL1-CAG93 were harvested 48 h aftertransfection. The cells were washed in ice cold PBS, scraped andpelleted by centrifugation (2000× g, 10 min, 4° C.). Cells were lysed onice for 30 min in 500 ml lysis buffer [50 mM Tris/HCl (pH 8.8), 100 mMNaCl, 5 mM MgCl₂, 0.5% (w/v) NP-40, 1 mM EDTA] containing the proteaseinhibitors PMSF (2 mM), leupeptin (10 μl/ml), pepstatin (10 μg/ml),aprotinin (1 μg/ml) and antipain (50 μg/ml). Insoluble material wasremoved by centrifugation for 5 min at 14000 rpm in a microfuge at 4° C.Pellets containing the insoluble material were resuspended in 100 mlDNase buffer [20 mM Tris/HCl (pH 8.0), 15 mM MgCl₂], and DNase I(Boehringer Mannheim) was added to a final concentration of 0.5 mg/mlfollowed by incubation at 37° C. for 1 h. After DNase treatment theprotein concentration was determined by the Dot Metric assay (GenoTechnology) using BSA as a standard. Eight μl 1 M Tris/HCl (pH 8.4), 1μl 1% (w/v) SDS, 1 μl 0.2 M CaCl₂ and 10 μl trypsin (0.25 mg/ml) werethen added, and the mixture was incubated for an additional 4 h at 37°C. Digestions were terminated by adjusting the mixtures to 20 mM EDTA,2% (w/v) SDS and 50 mM DTT, followed by heating at 98° C. for 5 min.FIG. 9C shows that insoluble protein aggregates are being formed intransfected COS cells expressing the HD exon 1 protein with 51 and 93glutamines but not in COS cells expressing the normal exon 1 allele with20 glutamines or in the non-transfected control cells. Thus, as observedin vitro with purified GST fusion proteins, formation of high molecularweight protein aggregates in vivo occurs in a repeat length-dependentway and requires a polyglutamine repeat in the pathological range. Inaddition, like the in vitro aggregates, the HD exon 1 aggregates formedin vivo are resistant to digestion with trypsin as well as to boiling in2% (w/v) SDS.

EXAMPLE 7

Isolation of Amyloid-Like Protein Aggregates from Alzheimer's DiseaseBrain

It has been shown that the neurodegenerative-disorder Alzheimer'sdisease (AD) is caused by the the formation of β-amyloids andneurofibrillar tangles (NFTs) mainly occuring in the neocortex,hippocampus and amygdala (K. Beyreuther, and C. L. Masters, Nature 383,476 (1996)). To determine whether these structures can be detected bythe dot-blot filter retardation assay brain extracts of patients andcontrols were prepared and analyzed using the anti-Tau, anti-β-amyloidand anti-HD1 antibodies.FIG. 12 shows that with the anti-Tau and anti-β-amyloid antibodies NFTsand β-amyloids were detected in brain extracts prepared from patients A2and A3, but not in brain extracts prepared from patient A1 and thecontrols. Clinical studies revealed that the patients A2 and A3 hadAlzheimer's disease with an intermediate and severe intellectualimpairment, respectively, whereas patient A1 suffered only from moderateintellectual impairment. This indicates that the results obtained withthe filter retardation assay correlate with the severity of the disease.Using the HD1 antibody in the brain extracts prepared from AD patientsand controls no aggregated huntingtin protein was detected. However, theantibody reacted with the GST-HD51 protein which was used as a positivecontrol.Human cerebral cortex (−500 mg) was homogenized in 2.5 ml of lysisbuffer (0.32 M sucrose, 1 mM MgCl₂, 5 mM KH₂PO₄, pH 7.0, 1 mM PMSF)using nine strokes of a glass homogenizer. The homogenat was centrifugedfor 15 min at 500×g to remove the nuclei. The original supernatant wasthen centrifuged at 93500×g for 1 h yielding a membrane pellet. Thepellet was dissolved in 2–5 ml 100 mM Tris-HCl (pH 8), 0.5% SDS andtrypsin (Boehringer Mannheim, sequencing grade) was added to a finalconcentation of 0.05 mg/ml followed by incubation at 37° C. overnight.Digestions were terminated by adjusting the mixtures to 2% SDS and 50 mMDTT, followed by heating at 98° C. for 5 min. The mixture wascentrifuged for 1 h at 110000×g and the resulting pellet was resuspendedin 100 μl of water. Aliquots (2–10 μl) were then used for the analysiswith the dot-blot filter retardation assay.

EXAMPLE 8

Dot-Blot Filter Retardation Assay

The filter assay used to detect polyglutamine-containing huntingtinprotein aggregates has been described (hereinabove and in E.Scherzinger, R. Lurz, M. Trumaine, L. Margiarini, B. Hollenbach, R.Hasenbank, G. P. Bates, S. W. Davies, H. Lehrach, and E. E. Wanker, Cell90, 549 (1997)). Denatured and reduced protein samples were prepared asdescribed above, and aliquots corresponding to 50–250 ng fusion protein(GST-HD20DP and GST-HD51 DP) or 5–30 μg extract protein (pelletfraction) were diluted into 200 μl 0.1% SDS and filtered on a BRL dotblot filtration unit through a cellulose acetate membrane (Schleicherand Schuell, 0.2 μm pore size) that had been preequilibrated with 0.1%SDS. Filters were washed 2 times with 200 μl 0.1% SDS and were thenblocked in TBS (100 mM Tris/HCl, pH 7.4, 150 mM NaCl) containing 3%nonfat dried milk, followed by incubation with the anti-HD1 (1:1000)(see above and E. Scherzinger, R. Lurz, M. Trumaine, L. Margiarini, B.Hollenbach, R. Hasenbank, G. P. Bates, S. W. Davies, H. Lehrach, and E.E. Wanker, Cell 90, 549 (1997), the anti-Tau (Dako, 1:1000) or theanti-β-amyloid antibody (Dako, 1:300). The filters were washed severaltimes in TBS, then incubated with a secondary anti-rabbit or anti-mouseantibody conjugated to horse raddish peroxidase (Sigma, 1:5000) followedby ECL (Amersham) detection. The developed blots were exposed forvarious times to Kodak X-OMAT film or to a Lumi-imager (BoehringerMannheim) to enable quantification of the immunoblots. For detection andquantification of polyglutamine-containing aggregates generated from theprotease-treated fusion proteins GST-HD20DPBio and -HD51DPBio, thebiotin/streptavidin-AP detection system was used. Following filtration,the cellulose acetate membranes were incubated with 1% (w/v) BSA in TBSfor 1 h at room temperature with gentle agitation on a reciprocalshaker. Membranes were then incubated for 30 min withstreptavidin-alkaline phosphatase (Promega) at a 1:1000 dilution in TBScontaining 1% BSA, washed 3 times in TBS containing 0.1% (v/v) Tween 20and 3 times in BSA, and finally incubated for 3 min with either thefluorescent alkaline phosphatase substrate AttoPhos™ or thechloro-substituted 1,2-dioxetane chemiluminescence substrate CDP-Star™(Boehringer Mannheim) in 100 mM Tris/HCl, pH 9.0, 100 mM NaCl and 1 mMMgCl₂. Fluorescent and chemiluminescent signals were imaged andquantified with the Boehringer Lumi-Imager F1 system and LumiAnalyst™software (Boehringer Mannheim).

EXAMPLE 9

Microtitre Plate Filter Retardation Assay

To process a large number of proteolytic digestion reactions inparallel, a microtitre plate filter retardation assay was developed. Inthis assay a 96-well microtitre plate containing a cellulose acetatemembrane with a pore size of 0.45 mm (Whatman Polyfiltronics) was usedfor the retention of polyglutamine-containing protein aggregates.The following experimental protocol was employed:First, 15 μl GST fusion protein solution (200 μg/ml GST-HD51DPBio orGST-HD20DPBio in buffer P [20 mM Tris/HCl (pH 8.0), 150 mM NaCl]) and 15μl trypsin solution (10 μg/ml trypsin (Boehringer Mannheim, sequencinggrade) in buffer P) were combined in a 96-well Thermo-Fast®96 tube plate(Advanced Biotechnologies LTD) using a multi channel pipette(Eppendorf), and the microtitre plate was incubated for 16 hours at 37°C. Then 30 μl SDS/DTTsolution (4% SDS, 100 mM DTT in buffer P) wereadded to each well, the plate was sealed with a microtitre plate sealer(Biostat LTD) and the plate was heated in a 96-well MasterCycler(Eppendorf-Netheler-Hinz) for 5 min at 98° C.The sealing was removed and 50 μl of the reaction mix were transferredinto each well of a new 96-well microtitre plate containing a 0.45 μmcellulose acetate membrane, pre-equilibrated with 0.1% (w/v) SDS, usinga multi channel pipette. For equilibration of the cellulose acetatemembrane, the microtitre plate was placed into the QIAvac Manifold-96(Qiagen) and 200 μl 0.1% SDS was pipetted into each well of the plate.Vacuum was then applied until the SDS solution had passed through thefilter. Prior to addition of the protein solution, each well of thefilter plate was preloaded with an additional 200 μl of 0.1% SDS. Thediluted protein solution was then filtered through the membrane byapplying vacuum.The filterplate was washed with 2×200 μl 0.1% SDS and 2×200 ml TBS (100mM Tris/HCl (pH 7.4), 150 mM NaCl). Vacuum was used to remove washsolutions from the membrane. 200 μl 0.2% (w/v) BSA in TBS were pipettedinto each well of the filterplate, and the plate was incubated for 1 hat room temperature (RT) (blocking). Blocking buffer was removed bypipetting.Next, 200 μl streptavidin alkaline phosphatase (1:1000, Promega) in 0.2%(w/v) BSA/TBS were added to each sample, and the filterplate wasincubated for 1 h at RT. Streptavidin AP buffer was removed bypipetting. The filterplate was washed with 3×200 μl TTBS [100 mMTris/HCl (pH 7.4), 150 mM NaCl, 0.1% (v/v) Tween 20] and 3×200 μl TBS.Vacuum was used to remove wash solutions.200 μl detection buffer (50 mM Tris/HCl (pH 9.0), 500 mM NaCl, 1 mM MgCl₂) were added to each sample, incubated for 1 min and vacuum wasapplied to remove the buffer. 200 μl Attophos™ (10 mM AttoPhos™) indetection buffer were pipetted into each well of the filterplate,incubated for 1 h at RT, vacuum was applied to remove the buffer, andthe fluorescence emission of each well was measured with theCytoFluor®4000 (Perseptive Biosystems) at 485+/−20 (excitation) and530+/−25 (emission). Finally, the resultant images were analysed withCytoFluor 4.1 software and MS Excel 7.0.As expected from the text set of experiments, using fusions of GST andthe full-length HD exon 1 protein, only the cleavage products ofGST-HD51 DP were retained by the filter and were detected by thehuntingtin-specific antibody HD1, indicating the formation of highmolecular weight HD51DP aggregates from this fusion protein. Scanningelectron microscopy of the material retained on the surface of themembrane revealed bunches of long fibrils or filaments (FIG. 9B), whichwere not detected after filtration of the uncleaved GST-HD51DPpreparation or the protease-treated GST-HD20DP preparation. Theseresults indicate that an elongated polyglutamine sequence but not theproline rich region in the HD exon 1 protein is necessary for theformation of high molecular weight protein aggregates in vitro.

Essentially, the same results as with the dot blot filter retardationassay were obtained when the fusion proteins GST-HD20DPBio and-HD51DPBio were analysed with the microtitre plate filter retardationassay, indicating that this assay can be used for the high throughputisolation of chemical compounds from chemical libraries (FIGS. 11A and11B).

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1. A composition comprising (a) a nucleic acid molecule encoding afusion protein comprising (aa) a glutathione S-transferase (GST)(poly)peptide that enhances solubility and/or prevents aggregation ofsaid fusion protein; and (ab) a huntingtin (poly)peptide that has theability to self-assemble into fibrils or protein aggregates, whereinconnection of polypeptides (aa) and (ab) is via a linker or by a directattachment, and wherein at least one of the linker, (poly)peptide (aa)and (poly)peptide (ab) includes a cleavable site, and wherein saidhuntingtin (poly)peptide self-assembles subsequent to release from saidfusion protein, (b) a vector containing the nucleic acid molecule of(a); (c) a host transformed with the vector of (b); and/or (d) a fusionprotein encoded by the nucleic acid of (a).
 2. The composition of claim1 wherein the huntingtin (poly)peptide comprises a polyglutamineexpansion.
 3. The composition of claim 2 wherein said polyglutamineexpansion comprises at least 35 glutamines.
 4. The composition of claim3 wherein said polyglutamine expansion comprises at least 51 glutamines.5. The composition of claim 1 wherein said nucleic acid is DNA.
 6. Thecomposition of claim 1 wherein said vector is an expression vector or agene targeting vector.
 7. The composition of claim 1 wherein said hostis a bacterial cell, an animal cell, an insect cell, a plant cell, afungal cell, or a Pichia pastoris cell.
 8. The composition of claim 7wherein the bacterial cell is an E. coli cell.
 9. The composition ofclaim 7, wherein the animal cell is a mammalian cell.
 10. Thecomposition of claim 7, wherein the fungal cell is a yeast cell.
 11. Thecomposition of claim 10, wherein the yeast cell is a Saccharomyces orAspergillus cell.
 12. The composition of claim 1, wherein the huntingtin(poly)peptide consists of a huntingtin (poly)peptide encoded by thenucleic acid sequence of huntingtin exon 1 and includes a polyglutamineexpansion that comprises at least 35 glutamines.
 13. The composition ofclaim 12, wherein the polyglutamine expansion comprises at least 51glutamines.